Mini prep plasmid extraction kit

Bacterial colonies were isolated from a cooling tower on R2A agar and re-streaked three times to ensure pure cultures. Glycerol stock (15% glycerol in R2A medium) cultures were made for each strain for downstream applications. The identities of morphologically different colonies were determined by sequencing the 16S rRNA gene. Briefly, DNA was extracted from pure cultures using the Wizard genomic DNA purification Kit (Promega). The 16S rRNA gene was amplified by PCR, using bacterial primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). The PCR product was cloned into the pGEM-T Easy vector system (Promega). Clones were selected by blue white screening. Plasmids containing the 16S rRNA insert were extracted using a Miniprep plasmid extraction kit (QIAGEN). The insert was sequenced by Sanger sequencing at the Plateforme Génomique de l’Université Laval, Canada. The sequence was then analysed using NCBI BLAST. One of the isolates of interest showed 99.51% identity with Brevundimonas sp. strain HES1 (Accession MN081030.1). We named the strain Brevundimonas SPF441.

Due to its strong discriminatory power in identifying S. aureus strains, the thermonuclease (nuc) gene was chosen as the candidate gene for S. aureus detection (28 (link)), and the mecA and nuc genes were utilized as target genes for MRSA identification. The nuc and mecA gene sequences of MRSA strains were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). Using MEGA 7 software, several nuc and mecA gene sequence alignments were carried out. Following sequence alignment, corresponding specific sgRNAs were designed based on the conserved sequences of the nuc and mecA genes that matched the Cas12a protospacer adjacent motif (5′-TTTN) (see Table S1 in the supplemental material). sgRNAs were subsequently synthesized by TaKaRa Bio (Beijing, China). Using the Basic Local Alignment Search Tool (BLAST [http://blast.ncbi.nlm.nih.gov/Blast.cgi]), the specificity of the candidate target sequences of the nuc and mecA genes was determined, as was sequence alignment. In addition, partial sequences of the nuc and mecA genes were synthesized by Sangon Biotech (Shanghai, China), cloned into pET-28a vectors, and transformed into TOP10 competent cells. Recombinant pET-28a vectors harboring the nuc or mecA gene were extracted using a miniprep plasmid extraction kit (Qiagen, Hilden, Germany).

Regulatory regions (R1 and R2), identified by integration of previously published human islet ATAC-seq and H3K27ac ChIP-seq datasets [11 (link)], were PCR-amplified from genomic DNA extracted from EndoC-βH3 cells with primer sets (Table S5) designed by Primer3-based software and cloned into pGL4.23[luc/minP] vector (Promega) between KpnI and XhoI restriction enzyme sites. Plasmid DNA was extracted using mini-prep plasmid extraction kit and/or Maxi-prep plasmid extraction kit (QIAGEN). The constructs were further confirmed by the Sanger sequencing.